Study of the interrelationship between Legionella pneumophila and free-living amoeba under the condition of natural water temperature to solving legionaires' disease

通过培养法、实时PCR（RTPCR）和EMA处理实时PCR（EMA RTPCR）相结合，并通过透射电镜观察，证实了嗜肺乳杆菌在15℃条件下在棘阿米巴castellanii细胞内进入VNC（Viable but not culable）模式。另一方面，多食棘阿米巴和Haltomannella vermiformis未观察到VNC状态的转变。在东京地区两条主要河流的固定采样点对河水进行定期调查，未发现可培养的嗜肺乳杆菌细胞。结合RTPCR和EMA RTPCR检测到少量活菌，但活细胞数量基本相同，与季节无关。PCR法检测游离变形虫（FLA），取样点以Haltomanella属居多，未检出castellanii属。与嗜肺乳杆菌的相互关系受水温影响的FLA物种的缺失或河流流量较少遇到宿主阿米巴虫的机会可能是导致这一结果的原因。

It was demonstrated that L. pneumophila shifted into a VNC(Viable but not Culturable) mode inside the Acanthamoeba castellanii cells at a temperature of 15 degrees C by the combination of cultural method, real time PCR(RTPCR) and EMA treated real time PCR(EMA RTPCR), and by transmission electron microscope observation. On the other hand, the shift into VNC state was not observed in Acanthamoeba polyphaga and Haltomannella vermiformis. Culturable L. pneumophila cells were not detected in periodic survey of river water at fixed-sampling points targeting two major rivers in the Tokyo area. A few viable Legionella cells were detected by the combination of RTPCR and EMA RTPCR, however the numbers of viable cells were approximately-same without relation to season. The detection of free-living amoeba(FLA) by PCR showed that the genus Haltomanella was predominant in the sampling points and A. castellanii was not detected. The absence of FLA species that the interrelationship with L. pneumophila is affected by water temperature or few chances to encounter host amoeba by flow of the river might be cause of this result.